Interactions of gamma-aminobutyric acid (GABA) with benzodiazepines have been studied electrophysiologically and biochemically in rats. Benzodiazepines enhance GABA inhibition of firing in the dorsal raphe nucleus but are inactive by themselves. Agents such as amino-oxyacetic acid (AOAA) which raise GABA levels in vivo lead to a potentiation of benzodiazepine inhibition. These effects are blocked by (plus) bicuculline and picrotoxin. Pretreatment of animals with AOAA and muscimol (a GABA agonist) in vivo lead to enhanced binding of (3H)diazepam measured in vitro. This enhanced binding is due to enhanced affinity of a specific benzodiazepine binding site in brain but not change in the number of sites. Including GABA or muscimol in direct (3H)diazepam binding assays in vitro also leads to enhanced binding. This effect is stereospecifically blocked by bicuculline and provides direct evidence for a GABA-benzodiazepam interaction.